Evaluation of the efficacy of creatine chemical exchange saturation transfer imaging in assessing testicular maturity

Abstract Purpose Microscopic testicular sperm extraction is the most effective treatment for NOA, but the sperm retrieval rate is low and depends on testicular maturity. However, there are limited useful tests to assess testicular maturity. Chemical exchange saturation transfer (CEST) imaging is a new magnetic resonance imaging (MRI) technique that can image the distribution of trace substances in vivo. We focused on the potential role of creatine (Cr) in testes and hypothesized that Cr‐CEST could indicate intratesticular spermatogenesis. Methods We performed Cr‐CEST by using 7T MRI on wild‐type C57B6/J mice and several types of male infertility models such as Sertoli‐cell only (SCO) (Kitw/Kitwv), maturation arrest (MA) (Zfp541 knockout mouse and Kctd19 knockout mouse), and teratozoospermia (Tbc1d21 knockout mouse). After performing Cr‐CEST, histological analysis was performed. Results The SCO and MA models showed decreased CEST signal intensity (p < 0.05), while no reduction was observed in the teratozoospermia model (p = 1.0). CEST signal intensity increased as the spermatogenesis stage progressed from the SCO model to the MA and teratozoospermia models. Furthermore, CEST signal intensity was reduced in 4‐week‐old wild‐type mice with immature testes (p < 0.05). Conclusions This study suggests that Cr‐CEST evaluates intratesticular spermatogenesis noninvasively and provides a new therapeutic strategy for treating male infertility.


| INTRODUC TI ON
While the global human population is on the rise, 1 the birthrate is declining in the United States, China, and other major countries, 2 which is becoming a problem. Infertility is a cause of declining fertility, with one in five to six couples who wish to conceive being infertile, and male infertility accounts for 50%. 3 Semen analysis is performed as a test for male infertility, and 1%-3% have azoospermia; the absence of sperm in the semen. 4 Azoospermia is the most severe form of male infertility, with approximately 20% of cases being obstructive azoospermia, in which the spermatic tract, including the vas deferens, is obstructed. Most of the remaining cases are nonobstructive azoospermia (NOA), in which testicular function is impaired. 4 Patients with NOA are commonly treated with microscopic testicular sperm extraction (micro-TESE), 5,6 which is currently the most effective sperm retrieval method. However, the sperm retrieval rate is only approximately 30% and the procedure is highly invasive, often resulting in suffering for many patients. 4,7 The low sperm retrieval rate is attributed to a lack of preoperative tests for predicting intratesticular maturity, and the development of new tests is a major unmet need.
MRI is the imaging of protons using a magnetic field, and most protons in vivo are derived from water and fat. Therefore, MRI is an imaging of water and fat; however, evaluation of molecules at low concentrations other than water and fat has been difficult so far.
Chemical exchange saturation transfer (CEST) imaging is a method of estimating the concentration of target low-concentration molecules, such as creatine, by irradiating them with specific electromagnetic waves. 8 CEST imaging has been increasingly reported in recent years, including APT-CEST for amide protons, and glutamine CEST (Glu-CEST) for glutamine. APT-CEST has already been clinically applied for brain tumor grading, 9,10 and Glu-CEST is expected to be used for functional imaging of nerves. 11 Testicular creatine (Cr) levels are the second highest in the body after skeletal muscle, 12 and it is known that Cr synthase knockout mice are infertile 13 and that Cr in the testes is decreased in patients with Klinefelter syndrome. 14 Focusing on Cr in the testes, we have already successfully measured Cr in the testes using creatine CEST (Cr-CEST) ( Figure 1A). [15][16][17] We have evaluated testicular injury models such as testicular ischemia, 15 irradiation, 16 and administration of anticancer drugs 17 ( Figure 1B) Kctd19 KO mice were used as models for maturation arrest (MA) and were generated using the methodology previously described in Ref. 20 Kctd19 KO mice exhibited typical metaphase I-arrested phenotype. In addition, ZFP541 KO mice were generated as a model in which differentiation stops early in meiosis. Tbc1d21 KO mice were used as a model of teratozoospermia and were generated using the methodology previously described in Ref. 19 Kit w /Kit wv , Zfp541 KO, and Kctd19 KO mice underwent MRI imaging at 4 weeks of age because their testes atrophy considerably with age. Tbc1d21 KO mice were imaged at 40 weeks of age. The CEST and the T2-weighted images obtained by S0 were superimposed to create a figure ( Figure 1C).

| Data processing
All image processing and data analysis was performed with in-house scripts written in MATLAB R2017b (MathWorks). S Xppm is defined as the signal intensity obtained by sequence with saturation pulse at Xppm. The CEST signal intensity was evaluated using magnetization transfer ratio (MTR) asymmetry analysis, determined from the following equation: The Cr-CEST effect was evaluated at 1.8 ppm based on our previous study. [15][16][17] MTR asym (Xppm) = S −xppm − S xppm ∕ S0

| Histological examination
Testes were harvested and fixed in 10% formalin fixative. The tissues were embedded in paraffin wax, sectioned at 5 μm, stained with hematoxylin and eosin (H&E), and examined using a keyence BZ-X700 microscope (Keyence, Co).

| Statistical analysis
All data were analyzed using JMP® 15 (SAS Institute Inc.). All data are presented as the mean ± standard error of the mean (SEM), and p-values < 0.05 were considered statistically significant. Twotailed Student's t-test for experiments with two groups and the Tukey-Kramer method for experiments including ≥3 groups were used for analysis as appropriate. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

| Zfp541 KO mice showed male infertility
Zfp541 is a gene specifically expressed in the testes and is known to stop sperm maturation at the pachytene stage. 20 Zfp541 KO mice showed infertility and a marked decrease in testicular weight ( Figure 2D-F). PAS staining showed no round spermatocytes, suggesting that sperm maturation ceased at the pachytene stage ( Figure 2G). In the cauda epididymis, no spermatozoa were observed ( Figure 2G).

| Evaluation of testicular pathology in male infertility models
In the present study, we used Kit w /Kit wv mice as a model of SCO, the most severe type of NOA. Zfp541 KO and Kctd19 KO mice were used as models of MA, which is a less severe type of NOA than SCO. Zfp541 KO and Kctd19 KO are used as models of maturation arrest in early-first (pachytene phase) and mid-first meiosis (metaphase 1), respectively. Tbc1d21 KO mice were used as a model of teratozoospermia. In this model, sperm were formed in the testes; however, mitochondrial sheath dysplasia occurred, resulting in infertility.
Kit w /Kit wv mice had no germ cells in the seminiferous tubule and markedly smaller testes ( Figure 3A). Zfp541, Kctd19 KO mice showed germ cells in the seminiferous tubule but no sperm formation ( Figure 3A). The testes of Zfp541 KO and Kctd19 KO were larger than those of Kit w /Kit wv but smaller than those of the wild type.
Tbc1d21 KO mice had spermatozoa in the seminiferous tubule and no testicular atrophy ( Figure 3A).  Figure 3A). There was no significant difference between the two strains (p = 1.0, Figure 3A). Contrastingly, when compared to the SCO model, the signal intensity ratio was significantly increased in the MA model (Zfp541 KO mice vs. Kit w /Kit wv mice: p = 0.017, Kctd19 KO mice vs.

| Cr-CEST signal intensity decreased in NOA models
Kit w /Kit wv mice: p = 0.025, Figure 3B). As a preliminary study, we compared signal intensity and intratesticular Cr levels measured by colorimetric assay in 4 weeks old Kit w /Kit wv and WT mouse (n = 1).
Cr concentration of the Kit w /Kit wv mouse was also considerably lower compared to WT mouse ( Figure S1).

| Cr-CEST signal intensity did not decrease in the teratozoospermia model
No decrease in CEST signal intensity was observed in Tbc1d21 KO mice (Control vs. Tbc1d21 KO mice: 6.5 ± 0.34 vs. 6.3 ± 0.33, Figure 3A). Furthermore, Tbc1d21 KO mice showed a significantly increased signal intensity ratio compared to the SCO and MA models ( Figure 3B). Based on the histopathology image and Cr-CEST signal, we considered that the signal intensity depends on the cell density. A comparison of cell density between Tbc1d21 mice and SCO-and MA-model mice showed that cell density was significantly increased in Tbc1d21 mice ( Figure 3C).

F I G U R E 3
Comparison of histopathological image of male infertility models. (A) Cr-CEST images and histopathology in the Sertoli-cellonly, maturation arrest, and teratozoospermia models, and comparison of signal intensity between each model and the wild type (WT) or hetero (scale bar = 50 μm). Data are expressed as the mean ± SEM (n = 3 mice per group). Statistical analysis was performed using the twotailed Student's t-test. (B) Comparison of CEST signal intensity ratio for each model. The signal intensity ratio is the ratio of each group to the control. Statistical analysis was performed using the Tukey-Kramer method. (C) Comparison of cell density ratio for each model. The signal intensity ratio is the ratio of each group to the control. Statistical analysis was performed using the Tukey-Kramer method.

| DISCUSS ION
This study found that Cr-CEST was an effective new method for assessing testicular maturity. Cr-CEST signal intensity was reduced in SCO and MA models, similar to previous testicular damage models. 15,16 In the teratozoospermia model, Cr-CEST signal intensity was equivalent to hetero mice with normal testicular findings. Furthermore, CEST signal intensity increased as the spermatogenesis stage progressed from the SCO model to the MA and teratozoospermia models. Similarly, Cr-CEST signal intensity in WT mice was significantly lower at 4 weeks of age (before sperm maturation was complete) than at 9-20 weeks of age (after sperm maturation was complete). This indicated that Cr-CEST, that is, Cr concentration in the testes, is a suitable indicator of sperm maturation in the testes.
Cr is converted to creatine phosphate (PCr) by creatine kinase, and PCr is stored as an energy-storage substance. In muscles, PCr is used during ATP production during exercise. Therefore, in muscle, PCr is much more abundant than Cr. 12 On the other hand, in the testes, which have the second highest total Cr content in the body after skeletal muscle, it is not present as PCr and is mostly retained as Cr. 24 This leads us to believe that Cr may have a role other than that of an energy-storage substance in the testis. Creatine synthase knockout mice are known to be infertile 13 ; however, the role of Cr in the testis is still not clear. In recent years, an increasing number of studies have focused on Cr, and it is known that Cr is involved in T-cell immunity, 25 that Cr is required for oligodendrocyte survival, 26 and that abnormalities in the Cr transporter are observed in patients with inflammatory bowel disease (IBD). 27 In patients with inflammatory bowel disease, Cr is reduced, causing a decrease in the tight junction function of the intestinal tract, and administration of Cr has reportedly improved symptoms, attracting attention as a novel treatment. 28 The testes also have a tight junction in the blood-testis barrier, and disruption of the tight junction leads to infertility. 29 We believe that if Cr is proven to be involved in this blood-testis barrier, it could be a novel treatment for male infertility.
In this study, the number of cells in the seminiferous tubules in the NOA models is reduced when compared to the WT, while the number of cells in the seminiferous tubules is not reduced in the teratozoospermia model because the maturation of the testes is normal. Therefore, Cr concentration in the testes may reflect cell density. In the present study, Cr-CEST signal intensity in infertility model mice was heterogeneous in the testes, and we hypothesized that there might be a correlation between the areas of high signal intensity and cell density. Therefore, we compared the histopathology and Cr-CEST images, and found no clear correlation between the morphology and the Cr-CEST signal intensity. This could be due to the fact that Cr-CEST images are imaged in 1 mm slices and tissue images are sectioned in 5 μm slices, making it difficult to evaluate them in the same slice. We have already studied and reported on the spatial resolution of Cr-CEST. 16 We performed partial irradiation of only the lower half of the mouse testis, and compared to the nonirradiated area, the irradiated area showed a significant decrease in Cr-CEST signal intensity, consistent with the histopathology image.
In view of the high spatial resolution of Cr-CEST, the fact that the Cr-CEST signal intensity was heterogeneous within the testes may indicate that Cr concentrations are heterogeneous within the testes in the mouse used in this study. Although this study could not clarify the heterogeneity of Cr-CEST signal intensity in the testis, it is conceivable that differences in Cr concentration in the seminiferous tubules may have an important role in spermatogenesis, and further studies on the role of Cr in the testis are expected in the future.
NOA can be divided into SCO (no germ cells present in the testes), and MA (germ cells are present but not yet differentiated into spermatozoa). The sperm retrieval rate of micro-TESE for SCO is quite low, while the sperm retrieval rate for MA is increased with treatment. 30,31 The success rate of micro-TESE will increase if SCO and MA can be distinguished in preoperative examinations. In addition, NOA patients have heterogeneous maturity within the testes, and some patients have areas of normal sperm formation. 18 Considering that Cr-CEST can predict testicular maturity, the use of Cr-CEST as a preoperative navigation system could increase the sperm retrieval rate of micro-TESE and reduce unnecessary surgery. In the future, we would like to conduct clinical trials in humans and apply them to a navigation system for micro-TESE.
This study has several limitations. First, it was impossible to examine Cr-CEST in mice younger than 4 weeks because they could not tolerate prolonged anesthesia. Second, in the examination of the infertility models, we performed Cr-CEST at 4 weeks old, before maturity was complete. In the infertility models, the testes had atrophied considerably after 4 weeks old, making MRI imaging difficult; therefore, this study was conducted at 4 weeks old. Third, Cr-CEST was not applied to humans. Cr-CEST of mouse testes was imaged using 7T MRI; however, most MRIs used in humans are 3T. Since 7T is the most common MRI for animal experiments, it was not possible to conduct a study at 3T. Therefore, it is necessary to investigate the use of 3T MRI in the future.
In conclusion, this study showed that Cr-CEST is a useful and novel noninvasive method for assessing maturity in the testis. Tsuchiya and A. Yasumoto for technical assistance with the experiments. The author would like to also thank Editage for English proofreading.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflict of interest.

A N I M A L S TU D I E S
All animal procedures were approved by the Institutional Animal Care and Use Committee of Osaka University (J007559-005).